Effects of margarine and butter consumption on distribution of trans-18∶1 fatty acid isomers and conjugated linoleic acid in major serum lipid classes in lactating women

Trans FA (TFA) have at least one trans double bond and comprise several isomers and types, including many of the CLA (e.g., c9, t11–18∶2 CLA). Some TFA may have adverse effects (e.g., cardiovascular disease), whereas some are though to have beneficial effects (e.g., anticarcinogenicity). The presence of TFA in human tissues and fluids is related to dietary intake, although this relationship is not completely understood—especially in regard to serum lipid fractions. This study was conducted as part of an investigation designed to test the influence of butter (B), “low TFA” margarine (LT), and regular margarine (RM) on milk fat content. Here we tested the secondary hypothesis that consumption of B, LT, and RM by lactating women would result in differential distribution of TFA and CLA in major serum lipid classes. Breastfeeding women (n=11) participated in this randomized Latinsquare study consisting of five periods: intervention I (5 d), washout I (7 d), intervention II (5 d), washout II (7 d), and intervention III (5 d). Extracted serum lipid was separated into cholesterol ester (CE), TAG, and phospholipid (PL) fractions and analyzed for total and isomeric TFA and CLA concentrations. Data indicate that TAG consistently contained the highest concentration of total t-18∶1. No interaction between treatment and fraction was found for any of the t-18∶1 isomers identified. Absolute concentration of each t-18∶1 isomer was greatest during the RM period, regardless of fraction. On a relative basis, concentrations of t10–18∶1 and t12–18∶1 were most responsive to treatment in the CE fraction. The concentration of c9, t11–18∶2 CLA was highest in the TAG fraction and lowest in the PL fraction, regardless of treatment. In summary, these results indicate (i) that there is a differential distribution of some isomeric TFA and CLA among human serum lipid fractions and (ii) that dietary TFA intake influences absolute and relative concentrations of some of the isomers in selected fractions.     

Differences in CLA isomer distribution of cow's milk lipids

The uniqueness of ruminant milk lipids is based on their high concentration of CLA. Maximal CLA concentrations in milk lipids require optimal conditions of ruminal fermentation and substrate availability, conditions like those present in pasture-fed cows. Our previous work showed that farm management (indoor feeding vs. pasture feeding) markedly influenced the CLA concentration. In this study, the objective was to evaluate the influence of the farm management system as dependent on different locations. Milk samples from different locations (Thuringia and the Alps, representing diverse altitudes) were collected during the summer months and analyzed for FA profile and CLA isomer distribution. The proportion of PUFA and total CLA in milk fat was significantly lower in milk from indoor cows compared with the pasture cows in the Alps. The trans-11 18∶1 in milk fat of Alpine cows was elevated, in contrast to lower values for trans-10 18∶1. Milk from cows grazing pasture in the Alps was higher in EPA and lower in arachidonic acid than milk from indoor-fed cows. The proportion of cis,trans/trans,cis isomers of CLA was 10 times higher from the indoor cows than from the Alpine cows. In addition to the major isomer cis-9,trans-11, this difference also occurred for the trans-11,cis-13 isomer, which represented more than a fourth of the total CLA present in milk fat. This is the first report showing a special isomer distribution in the milk fat of cows living under very natural conditions. We hypothesize that the CLA isomer trans-11,cis-13 is formed in large quantity as a result of grazing mountain pasture, which is rich in α-linolenic acid.     

Changes in conjugated linolenic acid composition within samples obtained from a single source

Conjugated linoleic acid (CLA; 9c, 11t-18∶2) and CLA isomers have been reported, in animals, to exhibit a variety of health-related benefits. Silver ion high-performance liquid chromatography (Ag-HPLC) was found to provide better resolution of the isomes than gas chromatography. Most commercially available samples of CLA, prepared by base-catalyzed isomerization of linoleic acid (9c, 12c-18∶2), are conposed of mixtures of four major isomers. While these isomers have been characterized, we found significant changes in CLA isomer ratios within samples obtained from the same producer/commercial supplier over a period of 1.5 yr. In the first sample, the four cis/trans isomers (8t, 10c-18∶2, 9c, 11t-18∶2, 10t, 12c-18∶2 and 11c, 13t-18∶2) were present in a ratio of approximately 1∶2∶2∶1, while in the second sample they were present in almost equal proportions. If indeed certain daily levels of CLA intake are required to produce suggested health benefits in humans, changes in concentrations of specific CLA isomers could significantly impact these effects. Care must be taken to analyze the CLA used in human and animal studies.     

Trans fatty acid isomers in Canadian human milk

The fatty acid composition, totaltrans content (i.e., sum of all the fatty acids which may have one or moretrans double bonds) and geometric and positional isomer distribution of unsaturated fatty acids of 198 human milk samples collected in 1992 from nine provinces of Canada were determined using a combination of capillary gas-liquid chromatography and silver nitrate thin-layer chromatography. The mean totaltrans fatty acid content was 7.19±3.03% of the total milk fatty acids and ranged from 0.10 to 17.15%. Twenty-five of the 198 samples contained more than 10% totaltrans fatty acids, and thirteen samples contained less than 4%. Totaltrans isomers of linoleic acid were 0.89% of the total milk fatty acids with 18∶2Δ9c, 13t being the most prevalent isomer, followed by 18∶2Δ9c, 12t and 18∶2Δ9t, 12c. Using the totaltrans values in human milk determined in the present study, the intake of totaltrans fatty acids from various dietary sources by Canadian lactating women was estimated to be 10.6±3.7 g/person/d, and in some individuals, the intake could be as high as 20.3 g/d. The 18∶1trans isomer distribution differed from that of cow's milk fat but was remarkably similar to that in partially hydrogenated soybean and canola oils, suggesting that partially hydrogenated vegetable oils are the major source of thesetrans fatty acids.     

Effect of dietary conjugated linoleic acid (CLA) on metabolism of isotope-labeled oleic,linoleic, and CLA isomers in women

The purpose of this study was to investigate the effect of dietary CLA on accretion of 9c-18∶1, 9c, 12c-18∶2, 10t, 12c-18∶2, and 9c, 11t-18∶2 and conversion of these FA to their desaturated, elongated, and chain-shortened metabolites. The subjects were six healthy adult women who had consumed normal diets supplemented with 6 g/d of sunflower oil or 3.9 g/d of CLA for 63 d. A mixture of 10t, 12c-18∶2-d ₄, 9c, 11t-18∶2-d ₆, 9c-18∶1-d ₈, and 9c, 12c-18∶2-d ₂, as their ethyl esters, was fed to each subject, and nine blood samples were drawn over a 48-h period. The results show that dietary CLA supplementation had no effect on the metabolism of the deuterium-labeled FA. These metabolic results were consistent with the general lack of a CLA diet effect on a variety of physiological responses previously reported for these women. The ²H-CLA isomers were metabolically different. The relative percent differences between the accumulation of 9c, 11t-18∶2-d ₆ and 10t, 12c-18∶2-d ₄ in plasma lipid classes ranged from 9 to 73%. The largest differences were a fourfold higher incorporation of 10t, 12c-18∶2-d ₄ than 9c, 11t-18∶2-d ₆ in 1-acyl PC and a two- to threefold higher incorporation of 9c, 11t-18∶2-d ₆ than 10t, 12c-18∶2-d ₄ in cholesterol esters. Compared to 9c-18∶1-d ₈ and 9c, 12c-18∶2-d ₂, the 10t, 12c-18∶2-d ₄ and 9c, 11t-18∶2-d ₆ isomers were 20–25% less well absorbed. Relative to 9c-18∶1, incorporation of the CLA isomers into 2-acyl PC and cholesterol ester was 39–84% lower and incorporation of 10t, 12c-18∶2 was 50% higher in 1-acyl PC. This pattern of selective incorporation and discrimination is similar to the pattern generally observed for trans and cis 18∶1 positional isomers. Elongated and desaturated CLA metabolites were detected. The concentration of 6c, 10t, 12c-18∶3-d ₄ in plasma TG was equal to 6.8% of the 10t, 12c-18∶2-d ₄ present, and TG was the only lipid fraction that contained a CLA metabolite present at concentrations sufficient for reliable quantification. In conclusion, no effect of dietary CLA was observed, absorption of CLA was less than that of 9c-18∶1, CLA positional isomers were metabolically different, and conversion of CLA isomers to desaturated and elongated metabolites was low.     

Synthesis and isolation of <Emphasis Type="Italic">trans</Emphasis>-7, <Emphasis Type="Italic">cis</Emphasis>-9 octadecadienoic acid and other CLA isomers by base conjugation of partially hydrogenated γ-linolenic acid

CLA is of considerable interest because of reported potentially beneficial effects in animal studies. CLA, while not yet unambiguously defined, is a mixture of octadecadienoic acids with conjugated double bonds. The major isomer in natural products is generally considered to be cis-9,trans-11-octadecadienoic acid (c9, t11), which represents >75% of the total CLA in most cases. Other isomers are drawing increased attention. The t7,c9 isomer, which is often the second-most prevalent CLA in natural products, has been reported to represent as much as 40% of total CLA in milk from cows fed a high-fat diet. The need for a reference material became apparent in a recent study directed specifically at measuring t7,c9-CLA in milk, plasma, and rumen. A suitable standard mixture was produced by stirring 0.5 g of γ-linolenic acid (all cis-6,9, 12-C_18∶3) with 100 mL of 10% hydrazine hydrate in methanol for 2.5 h at 45°C. The solution was diluted with H₂O and acidified with HCl. The resulting partially hydrogenated FA were extracted with ether/petroleum ether, dried with Na₂SO₄, and conjugated by adding of 6.6% KOH in ethlylene glycol and heating for 1.5 h at 150–160°C. Approximately 20 mg each of cis-6, trans-8; trans-7, cis-9; cis-9, trans-11; and rans-10, cis-12 were obtained along with other FA. Methyl esters (FAME) of these four cis/trans isomers were resolved by Ag⁺HPLC (UV 233) and partially resolved by GC/(MS or FID) (CP-Sil 88). Treatment of these FAME with I₂ yielded all possible cis/trans (geometric) isomers for the four positions 6,8; 7,9; 9,11; and 10,12.     

β-Oxidation of conjugated linoleic acid isomers and linoleic acid in rats

To assess the oxidative metabolism of conjugated linoleic acid (CLA) isomers, rats were force-fed 1.5–2.6 MBq of [1-¹⁴C]-linoleic acid (9c,12c-18∶2),-rumenic acid (9c,11t-18∶2), or-10trans, 12cis-18∶2 (10t, 12c-18∶2), and ¹⁴CO₂ production was monitored for 24 h. The animals were then necropsied and the radioactivity determined in different tissues. Both CLA isomers were oxidized significantly more than linoleic acid. Moreover, less radioactivity was recovered in most tissues after CLA intake than after linoleic acid intake. The substantial oxidation of CLA isomers must be considered when assessing the putative health benefits of CLA supplements.     

Biosynthesis of conjugated linoleic acid in humans

This paper deals with the reanalysis of serum lipids from previous studies in which deuterated fatty acids were administered to a single person. Samples were reanalyzed to determine if the deuterated fatty acids were converted to deuterium-labeled conjugated linoleic acid (CLA, 9c, 11t-18∶2) or other CLA isomers. We found 11-trans-octadecenoate (fed as the triglyceride) was converted (Δ9 desaturase) to CLA, at a CLA enrichment ofca. 30%. The 11-cis-octadecenoate isomer was also converted to 9c, 11c-18∶2, but at <10% the concentration of the 11t-18∶1 isomer. No evidence (within our limits of detection) for conversion of 10-cis-or 10-trans-octadecenoate to the 10,12-CLA isomers (Δ12 desaturase) was found. No evidence for the conversion of 9-cis, 12-cis-octadecadienoate to CLA (via isomerase enzyme) was found. Although these data come from isomerase enzyme) was found. Although these data come from four single human subject studies, data from some 30 similar human studies have convinced us that the existence of a metabolic pathway in one subject may be extrapolated to the normal adult population.     

In vitro comparison of hepatic metabolism of 9cis-11 trans and 10trans-12cis isomers of CLA in the rat

Hepatic metabolism of the two main isomers of CLA (9cis-11 trans, 10trans-12cisC_18∶2) was compared to that of oleic acid (representative of the main plasma FA) in 16 rats by using the in vitro method of incubated liver slices. Liver tissue samples were incubated at 37°C for 17h under an atmosphere of 95% O₂/5%CO₂ in a medium supplemented with 0.75 mM of FA mixture (representative of circulating nonesterified FA) and with 55 μM [1-¹⁴C]9cis-11 trans C_18∶2, [1-¹⁴C]10trans-12cis C_18∶2, or [1-¹⁴C]oleate. The uptake of CLA by hepatocytes was similar for both isomers (9%) and was three times higher (P<0.01) than for oleate (2.6%). The rate of CLA isomer oxidation was two times higher (49 and 40% of incorporated amounts of 9cis-11 trans and 10trans-12 cis, respectively) than that of oleate (P<0.01). Total oxidation of oleate and CLA isomers into [¹⁴CO₂] was low (2 to 7% of total oxidized FA) compared to the partial oxidation (93 to 98%) leading to the production of [¹⁴C] acid-soluble products. CLA isoemrs escaping from catabolism were both highly desaturated (26.7 and 26.8%) into conjugated 18∶3. Oleate and CLA isomers were mainly esterified into neutral lipids (30%). They were slowly secreted as parts of VLDL particles (<0.4% of FA incorporated into cells), the extent of secretion of oleate and of 10trans-12 cis being 2.2-fold higher than that of 9cis-11 trans (P<0.02). In conclusion, this study clearly showed that both CLA isomers were highly catabolized by hepatocytes, reducing their availability for peripheral tissues. Moreover, more than 25% of CLA escaping from catabolism was converted into conjugated 18∶3, the biological properties of which remain to be elucidated.     

Changes in the milk and cheese fat composition of ewes fed commercial supplements containing linseed with special reference to the CLA content and isomer composition

A study was carried out to increase the CLA contents in ewes’ milk fat under field conditions by dietary means and to investigate the extent of the changes and consequences for milk processing and cheese quality. During a 3-mon period, ewes’ bulk milk samples were collected every week from two different herds. For the first 4 wk the ewes were fed a conventional diet. Then the following 6 wk a supplement enriched in α-linolenate (whole linseed) was incorporated into the ovine diet. Finally, in the last 3 wk the feeding was the same as in the first 4 wk. The FA profile in milk fat was monitored by GC, and the distribution of CLA isomers was thoroughly tested by combining GC-MS of 4,4-dimethyloxazoline derivatives (DMOX) with silver ion-HPLC (Ag⁺-HPLC) of FAME. Reconstructed mass spectral profiles of CLA characteristic ions from DMOX were used to identify positional isomers, and Ag⁺-HPLC was used to quantify them. An increase in total CLA in milk fat was observed, and total CLA remained elevated during the weeks of enriched α-linolenate feeding. In our experimental conditions there was a linear relationship between trans-vaccenic acid (trans-11-octadecenoic acid; trans-11 18∶1) and 9-cis, 11-trans CLA in ewes’ milk fat. Concerning the CLA isomer profile, increases in the 11,13- and 12,14–18∶2 positional isomers were considerable when linseed was included in the diet. Organoleptic characteristics of cheeses made with CLA-enriched milk did not substantially differ from those made with nonsupplemented ewes’ milk. CLA total content and isomer profile did not change during ripening.     

Identification of conjugated linoleic acid isomers in cheese by gas chromatography, silver ion high performance liquid chromatography and mass spectral reconstructed ion profiles. Comparison of chromatographic elution sequences

Commercial cheese products were analyzed for their composition and content of conjugated linoleic acid (CLA) isomers. The total lipids were extracted from cheese using petroleum ether/diethyl ether and methylated using NaOCH₃. The fatty acid methyl esters (FAME) were separated by gas chromatography (GC), using a 100-m polar capillary column, into nine minor peaks besides that of the major rumenic acid, 9c, 11t-octadecadienoic acid (18∶2), and were attributed to 19 CLA isomers. By using silver ion-high performance liquid chromatography (Ag⁺-HPLC), CLA isomers were resolved into seven trans, trans (5–9%), three cis/trans (10–13%), and five cis, cis (<1%) peaks, totaling 15, in addition to that of the 9c, 11t-18∶2 (78–84%). The FAME of total cheese lipids were fractionated by semipreparative Ag⁺-HPLC and converted to their 4,4-dimethyloxazoline derivatives after hydrolysis to free fatty acids. The geometrical configuration of the CLA isomers was confirmed by GC-direct deposition-Fourier transform infrared, and their double bond positions were established by GC-electron ionization mass spectrometry. Reconstructed mass spectral ion profiles of the m+2 allylic ion and the m+3 ion (where m is the position of the second double bond in the parent conjugated fatty acid) were used to identify the minor CLA isomers in cheese. Cheese contained 7 t,9c-18∶2 and the previously unreported 11t, 13c-18∶2 and 12c, 14t-18∶2, and their trans,trans and cis,cis geometric isomers. Minor amounts of 8,10-, and 10, 12–18∶2 were also found. The predicted elution orders of the different CLA isomers on long polar capillary GC and Ag^*-HPLC columns are also presented.     

Preparation, separation, and confirmation of the eight geometrical cis/trans conjugated linoleic acid isomers 8,10-through 11,13–18∶2

Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or I₂ catalyst. The resultant mixtures of the eight cis/trans geometric isomers of 8,10-, 9,11-, 10,12-, and 11,13-octadecadienoic (18∶2) acid methyl esters were separated by silver ion-high-performance liquid chromatography (Ag⁺-HPLC) and gas chromatography (GC). Ag⁺-HPLC allowed the separation of all positional CLA isomers and geometric cis/trans CLA isomers except 10,12–18∶2. However, one of the 8,10 isomers (8cis, 10trans-18∶2) coeluted with the 9trans,11cis18∶2 isomer. There were differences in the elution order of the pairs of geometric CLA isomers resolved by Ag⁺-HPLC. For the 8,10 and 9,11 CLA isomers, cis,trans eluted before trans,cis, whereas the opposite elution pattern was observed for the 11,13–18∶2 geometric isomers (trans,cis before cis,trans). All eight cis/trans CLA isomers were separated by GC on long polar capillary columns only when their relative concentrations were about equal. Large differences in the relative concentration of the CLA isomers found in natural products obscured the resolution and identification of a number of minor CLA isomers. In such cases, GC-mass spectrometry of the dimethyloxazoline derivatives was used to identify and confirm coeluting CLA isomers. For the same positional isomer, the cis,trans consistently eluted before the trans,cis CLA isomers by GC. High resolution mass spectrometry (MS) selected ion recording (SIR) of the molecular ions of the 18∶1 18∶2, and 18∶3 fatty acid methyl esters served as an independent and highly sensitive method to confirm CLA methyl ester peak assignments in GC chromatograms obtained from food samples by flame-ionization detection. The high-resolution MS data were used to correct for the nonselectivity of the flame-ionization detector.     

Maternal supplementation with CLA decreases milk fat in humans

CLA refers to isomers of octadecadienoic acid with conjugated double bonds. The most abundant form of CLA (rumenic acid (RA): c9,t11-18∶2) is found in milk and beef fat. Further, CLA supplements containing RA and t10,c12−18∶2 are now available. Consumption of commercially produced CLA has been shown to decrease adipose accretion in growing laboratory and production animals and cause milk fat depression in cows. We tested the hypothesis that CLA supplementation would increase milk CLA concentration and decrease milk fat content in humans. Breastfeeding women (n=9) participated in this double-blind, placebo-controlled, crossover study divided into three periods: intervention l (5 d), washout (7 d), and intervention II (5 d). Women were randomized to treatment order. During each intervention period, women consumed 1.5 g of CLA supplement or placebo (olive oil) daily; during the washout period, no supplements were consumed. Milk was collected by complete breast expression on the final day of each period; milk output was estimated by 24-h weighing on the penultimate day of each intervention period. Milk RA and t10,c12−18∶2 concentrations were greater (P<0.05) during the CLA treatment period as compared to the placebo period. Milk fat content was significantly lower during the CLA treatment, as compared to the placebo treatment (P<0.05). Data indicate no effect of treatment on milk output. Therefore, it would be prudent that lactating women not consume commercially available CLA supplements at this time. This paper was published in part in Masters, N., McGuire, M.A., and McGuire, M.K. (1999) Conjugated Linoleic Acid Supplementation and Milk Fat Content in Humans, FASEB J. 13, A697.     

Consumption of c9,t11-18:2 or t10,c12-18:2 enriched dietary supplements does not influence milk macronutrients in healthy, lactating women

Substantial research suggests that the t10,c12–18:2, but not the c9,t11–18:2, isomer of conjugated linoleic acid (CLA) reduces milk fat synthesis in lactating bovine and rodent species. Because fat is the major energy-yielding component in human milk, we were interested in whether this is true for women as well. Thus, the effects of c9,t11–18:2 and t10,c12–18:2 on milk fat were examined in breast-feeding women (n = 12) in a double-blind, placebo-controlled, crossover study with latin-square design. The study was divided into six periods: baseline (3 days), three intervention periods (5 days each), and two washout periods (9 days each). During each intervention period, women consumed 750 mg/day of a supplement containing predominantly c9,t11–18:2, t10,c12–18:2, or 18:1 (olive oil placebo). Milk was collected by complete breast expression on the final day of each period. Infant milk consumption was estimated by 24 h weighing on the penultimate day of each intervention and washout period, and maternal adiposity (% body fat) was determined at baseline using dual energy X-ray absorptiometry. Milk c9,t11–18:2 and t10,c12–18:2 concentrations were greater (P < 0.05) during the corresponding CLA treatment periods as compared to the placebo period, providing strong evidence of subject compliance. Both CLA isomers were transferred into milk fat at relatively high efficiency; average transfer efficiency was estimated to be 23.3%. Compared to the placebo treatment, milk fat content was not reduced during either CLA treatment. Data indicate that body fatness did not modify any putative effect of isomeric CLA consumption on milk fat concentration. The evidence from this study suggests that the sensitivity of lactating women’s mammary tissue to an anti-lipogenic effect of the t10,c12–18:2 isoform of CLA may be less than previously hypothesized.     

Effect of pasture vs. concentrate diet on CLA isomer distribution in different tissue lipids of beef cattle

This study examined the effects of feeding pasture vs. concentrate on the distribution of CLA isomers in the lipids of longissimus and semitendinosus muscle, liver and heart muscle, and subcutaneous fat in beef bulls. Sixty-four German Holstein and German Simmental bulls were randomly allocated to either an indoor concentrate system or periods of pasture feeding followed by a finishing period on a concentrate containing linseed to enhance their beef content of n−3 PUFA and CLA. The concentrations of CLA isomers in the different tissues were determined by GC and silver ion HPLC. The diet affected the distribution of individual CLA isomers in the lipids of the different tissues. The concentration (mg/100 g fresh tissue) of the most prominent isomer, cis-9,trans-11 18∶2, was increased up to 1.5 times in liver and heart tissue of bulls fed on pasture as compared with concentrate. However, no diet effect was observed for cis-9,trans-11 18∶2 in the lipids of longissimus muscle and subcutaneous fat. In all tissues, the second-most abundant CLA isomer in concentratefed bulls was trans-7,cis-9 18∶2. In contrast, trans-11,cis-13 18∶2 was the second-most abundant CLA isomer in all investigated tissue lipids of pasture-fed bulls. The concentration of the trans-11,cis-13 18∶2 isomer was up to 15 times higher in tissues of pasture-fed bulls as compared with concentrate-fed animals. Furthermone, diet affected the concentrations of the CLA trans,trans 18∶2 isomers. Pasture feeding significantly increased the concentrations of some trans,trans 18∶2 isomers as compared with concentrate, predominantly trans-12,trans-14 18∶2 and trans-11,trans-13 18∶2. Overall, pasture feeding resulted in significantly increased concentrations of the sum of CLA isomers in the lipids of longissimus, muscle, subcutaneous fat, heart and liver muscle of German Holstein and German Simmental bulls, but not in semitendinosus muscle.     

Occurrence of geometrical isomers of eicosapentaenoic and docosahexaenoic acids in liver lipids of rats fed heated linseed oil

Monotrans geometrical isomers of 20∶5 n−3 and 22∶6 n−3 were detected in liver lipid of rats fed heated linseed oil. The isomers were identified as being 20∶5 δ5c, 8c, 11c, 14c, 17t and 22∶6 δ4c, 7c, 10c, 13c, 16c, 19t. These fatty acids were isolated as methyl esters by preparative high-performance liquid chromatography (HPLC) on reversed phase columns followed by silver nitrate thin layer chromatography (AgNO₃-TLC). The structures were identified using partial hydrazine reduction, AgNO₃-TLC of the resulting monoenes, oxidative ozonolysis of each monoene band, and gas-liquid chromatography (GLC) of the resulting dimethyl esters and monomethyl esters. Fourier-transform-infrared spectrometry confirmed thetrans geometry in isolated 20∶5 and 22∶6 isomers. The isomers of eicosapentaenoic and docosahexaenoic acids in liver lipids probably resulted from desaturation and elongation of 18∶3 δ9c, 12c, 15t, a geometrical isomer of linolenic acid present in the heated dietary oil.     

Geometry of conjugated double bonds of CLA isomers in a commercial mixture and in their hepatic 20∶4 metabolites

Rats were fed a fat-free diet for 2 wk. After this period, while maintaining the animals on the same diet, the rats were given intragastrically 180 mg per day of a mixture of conjugated linoleic acids (CLA) as triacylglycerols. Gas chromatography-mass spectrometry (GC-MS) analyses of this mixture, as well as hydrazine reduction and GC-MS and GC-Fourier transform infrared analyses of the resulting monoenes, revealed the presence of two major isomers, the 9c, 11t-and the 10t, 12c-18∶2 accompanied by smaller amounts of the 8t, 10c and the 11c,13t−18∶2 isomers. Minor quantities of cis,cis and trans,trans conjugated isomers also were detected. The total fatty acid methyl esters from the liver lipids were fractionated by reversed-phase high-performance liquid chromatography, and the fraction containing the 20∶4 isomers was further fractionated by silver nitrate thin-layer chromatography. A band containing two 20∶4 conjugated isomers was submitted to hydrazine reduction and the resulting monoenes analyzed by GC-MS as dimethyl-oxazoline derivatives. The two conjugated isomers were tentatively identified as 5c,8c,11c,13t–20∶4 and 5c,8c,12t,14c−20∶4. These could be formed by desaturation and elongation of the 9c,11t-and 10t,12c−18∶2 present in the commerical CLA mixture.     

Milk fat synthesis is unaffected by abomasal infusion of the conjugated diene 18:3 isomers cis-6,trans-10, cis-12 and cis-6,trans-8,cis-12

It has been previously established that trans-10, cis-12 CLA is a potent inhibitor of milk fat synthesis. Although the mechanism of this action is not completely understood, it has been speculated that eicosanoid-like metabolites of this isomer formed by the activity of tissue desaturases may be responsible for its activity. The objective of this study was to investigate the effects of an enrichment containing an 18∶3 conjugated diene, produced in the metabolism of trans-10, cis-12 CLA, on milk fat synthesis. Three rumen-fistulated Holstein cows (210±8 d in milk) were randomly assigned in a 3×3 Latin square experiment. Treatments were (i) control, (ii) trans-10, cis-12 CLA supplement (2.1 g/d; positive control), (iii) enrichment providing two conjugated diene 18∶3 isomers (2.6 g/d of cis-6, trans-10, cis-12 and 4.0 g/d of cis-6, trans-8, cis-12) and trans-10, cis-12 CLA (2.1 g/d). Treatments were abomasally infused for 5 d at 4-h intervals, and there was a 7-d interval between periods. Milk yield, dry matter intake, and milk protein yield were unaffected by treatments. In contrast, the trans-10, cis-12 CLA supplement reduced milk fat yield by 27%, whereas the supplement enriched with conjugated diene 18∶3 isomers (treatment iii) had no effect on milk fat yield beyond that attributable to its trans-10, cis-12 CLA content. The transfer efficiency of trans-10, cis-12 CLA into milk fat was 25 and 24% for treatments ii and iii, respectively. At the same time, the abomasally infused conjugated diene 18∶3 isomers were transferred to milk fat with an efficiency of 33 and 41% for cis-6, trans-10, cis-12 and cis-6, trans-8, cis-12 18∶3, respectively. Overall, short-term abomasal infusion of the conjugated diene 18∶3 isomers had no effect on milk fat synthesis, thereby offering no support for an involvement of metabolies of trans-10, cis-12 CLA in the regulation of milk fat synthesis.     

Effect of CLA on the cellular lipids of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The yeast Saccharomyces cerevisiae was cultivated in the presence of free CLA that was either a pure trans-10, cis-12 isomer, a pure cis-9, trans-11 isomer, or a 1∶1 mixture of the two, and the influence of these supplementations on the content and FA composition of the lipids in the yeast was determined. Neither the pure isomers nor their 1∶1 mixture influenced the growth of the yeast, but the trans-10, cis-12 isomer reduced the amount of cellular lipids by 40%. The reduction in total cellular lipids by the trans-10, cis-12 CLA was due to a reduction in TAG. Both of the isomers were incorporated into the yeast lipids, reaching a proportion of about 33% in TAG. With the incorporation of CLA, the yeast reduced the amount and desaturation of endogenously synthesized FA. These clear and pronounced isomer-specific effects of CLA on the yeast suggest that yeast might be a useful model to obtain a more comprehensive view of the mechanisms of the action of CLA on lipid metabolism.     

Lipase-catalyzed fractionation of conjugated linoleic acid isomers

The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mixtures of conjugated linoleic acid (CLA) isomers during esterification of mixed CLA free fatty acids and during hydrolysis of mixed CLA methyl esters were examined. The enzymes were highly selective for cis-9,trans-11–18∶2. A commercial CLA methyl ester preparation, containing at least 12 species representing four positional CLA isomers, was incubated in aqueous solution with either a commercial G. candidum lipase preparation (Amano GC-4) or lipase produced from a cloned high-selectivity G. candidum lipase B gene. In both instances selective hydrolysis of the cis-9,trans-11–18∶2 methyl ester occurred, with negligible hydrolysis of other CLA isomers. The content of cis-9,trans-11–18∶2 in the resulting free fatty acid fraction was between 94 (lipase B reaction) and 77% (GC-4 reaction). The commercial CLA mixture contained only trace amounts of trans-9,cis-11–18∶2, and there was no evidence that this isomer was hydrolyzed by the enzyme. Analogous results were obtained with these enzymes in the esterification in organic solvent of a commercial preparation of CLA free fatty acids containing at least 12 CLA isomers. In this case, G. candidum lipase B generated a methyl ester fraction that contained >98% cis-9,trans-11–18∶2. Geotrichum candidum lipases B and GC-4 also demonstrated high selectivity in the esterification of CLA with ethanol, generating ethyl ester fractions containing 96 and 80%, respectively, of the cis-9,trans-11 isomer. In a second set of experiments, CLA synthesized from pure linoleic acid, composed essentially of two isomers, cis-9,trans-11 and trans-10,cis-12, was utilized. This was subjected to esterification with octanol in an aqueous reaction system using Amano GC-4 lipase as catalyst. The resulting ester fraction contained up to 97% of the cis-9,trans-11 isomer. After adjustment of the reaction conditions, a concentration of 85% trans-10,cis-12–18∶2 could be obtained in the unreacted free fatty acid fraction. These lipase-catalyzed reactions provide a means for the preparative-scale production of high-purity cis-9,trans-11–18∶2, and a corresponding CLA fraction depleted of this isomer.